Pyridyl hydrazones for the treatment of tuberculosis and related diseases

ABSTRACT

The invention provides pyridyl-hydfazones of Formula I, or pharmaceutically acceptable salts, esters, solvates, isprriers and prodrugs thereof as well as pharmaceutical compositions containing these compounds for use in the prophylactic and/or therapeutic treatment of tuberculosis and related diseases, such as, diseases caused by nontuberculous mycobacteria and/or caused by  Micobacterium leprae.

FIELD OF THE INVENTION

The invention relates to compounds and pharmaceutical compositions whichare useful as pharmaceutical agents for treating tuberculosis andrelated diseases.

BACKGROUND OF THE INVENTION

There are more than 120 members of the genus Mycobacterium, which arediverse in pathogenicity, in vivo adaptation, virulence, response todrugs and growth characteristics. Mycobacterial diseases are caused byorganisms of the Mycobacterium tuberculosis Complex (MtbC) likeMycobacterium tuberculosis (Mtb), Mycobacterium bovis, Mycobacteriumafricanum, Mycobacterium canetii and Mycobacterium microti. Mycobacteriaother than MtbC and Mycobacterium leprae are known as non-tuberculousmycobacteria (NTM) and can cause also human and animal diseases as isthe case for Mycobacterium avium complex (MAC), Mycobacterium smegmatis,Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium terrae,Mycobacterium scrofulaceum, Mycobacterium vaccae, Mycobacterium marinum,Mycobacterium lentiflavum, Mycobacterium fortuitum, Mycobacteriumchelonae and Mycobacterium abscessus.

Most human tuberculosis (TB) is caused by Mtb but some cases are due toMycobacterium bovis, which is the principal cause of tuberculosis incattle and many other mammals, or Mycobacterium africanum.

The consequences of tuberculosis on all human societies are dramatic:worldwide, one person out of three is infected with Mtb.

The lung is the main entrance gate of Mtb in the body and, consequently,TB is primarily a disease of the lungs. Mycobacterium tuberculosiscauses a focal infection in the site where it is deposited afterinhalation. If the infection cannot be contained at the local level,bacilli dissemination is produced initially by hematogenic route,probably inside phagocytic cells, towards different organs and,eventually, to the contiguous pleura. It reaches hilar lymph nodes viathe lymphatic route, and from there, a second systemic dissemination canoccur, through the thoracic duct and superior vena cava, with thedevelopment of local foci in the lungs. Extrapulmonary foci can also beproduced by hematogenic and lymphatic dissemination. Tuberculosis canproceed to a generalized infection (“miliary tuberculosis”).

The clinical manifestations of TB depend on the local organic defenseson the sites of bacilli multiplication. Primary TB infection occurscommonly during childhood and most of the times, causes no apparentsymptoms and stays latent for life or until reactivation. Occasionallycause malaise, low-grade fever, erythema nodosum and phlyctenularconjunctivitis. In seriously immunodepressed patients it can developinto a disseminated form, which is sometimes fatal. Miliary tuberculosisresults from the massive hematogenic dissemination of Mtb during theprimary infection.

The development of clinical TB will occur in 5%-10% of infected personsat some point in their lives. The existence of post-primary TB, alsoknown as secondary TB, means that the infection can progress after thedevelopment of an adequate specific immune response. This TB episode candevelop in two ways: by inhalation of new bacilli or by reactivation ofthe primary focus.

There are factors involved in increased risk of developing TB, of whichthe most important are those interfering directly with host immunity.Diseases and conditions that weaken immunity, such as malnutrition,alcoholism, illicit drug abuse, advanced age, human immunodeficiencyvirus (HIV) infection or acquired immunodeficiency syndrome (AIDS),diabetes mellitus, gastrectomy, chronic renal insufficiency, chronicliver disease, silicosis, paracoccidioidomycosis, leukemias, solidtumors, prolonged treatment with corticosteroids, immunosuppressive drugtreatments, organ transplant, systemic lupus erythematosus, treatmentwith anti-tumor necrosis factor (TNF) antibodies and hereditaryfeatures, are factors that facilitate the development of TB disease. Inindustrialized countries the increased survival rates have resulted inlarger elderly populations with an increased risk of reactivation of theinfection. Tuberculosis in the elderly may be due also to a newlyacquired infection. Congenital TB is considered a rare event in thewhole spectrum of TB presentations. This infection is caused bylymphohematogenous spread during pregnancy from an infected placenta oraspiration of contaminated amniotic fluid.

Additional factors include the infective bacterial load, virulence ofMtb and host genetic susceptibility.

Pulmonary TB is the most common form of post-primary disease. Thenatural evolution of post-primary lesions in immunocompetent persons canlead to dissemination and death in about 50% of cases, and to chronicityin about 25% to 30%.

After penetration into the organism through the respiratory route, Mtbcan multiply in any organ during the primary infection, beforedevelopment of the specific immune response. After this, tuberclebacilli can multiply at any time when there is a decrease in the host'simmune capacity to contain the bacilli in their implantation sites. Theextrapulmonary tuberculosis can affect any other organ of the body,including lymph nodes, pleura, genitourinary system, central nervoussystem, osteoarticular system, gastrointestinal system, skin and softtissues and eye.

Tuberculosis accounts for 2.5% of the global burden of disease and holdsthe seventh place in the global ranking of causes of death. In 2010,there were 8.8 million incident cases of TB, 1.1 million deaths from TBamong HIV negative people and an additional 0.35 million deaths fromHIV-associated TB. Without treatment, a person with active TB willinfect an average of 10 to 15 other people per year.

The minimum period of treatment for active, drug-sensitive TB is 6months, and will typically use a starting regimen of four drugsdenominated first-line drugs: isoniazid (INH), rifampicin (RMP),pyrazidamine (PZA) and ethambutol (EMB). However, when administered inreal-world settings, the regimen's flaws become apparent. Treatment ofdrug-resistant TB is even lengthier, taking 18-24 months or longer.

The selection of the drug regimen must be done considering at least thefollowing factors: disease localization and severity, result of sputumsmear microscopy, HIV co-infection, prevalence of drug resistance in thesetting, availability of drugs, cost of treatment and medicalsupervision, whether the patient has previously received anyanti-tuberculosis drug, the country's budget, health coverage by publichealth services and qualifications of health staff.

Human immunodeficiency virus infection has clearly had a profound effecton TB epidemiology. Human immunodeficiency virus infection is a potentrisk factor for TB and both form a lethal combination, each speeding theother's progress. Not only does HIV increase the risk of reactivatinglatent Mtb and the risk of rapid TB progression soon after Mtb infectionor reinfection. Those who have latent tuberculosis have a 10% lifetimerisk of progressing to active infection, with half (5%) occurring within1-2 years after initial infection. In persons co-infected with Mtb andHIV, however, the annual risk can exceed 10%.

The resistance mechanisms can be divided in natural and acquired. Thenatural drug resistance of Mtb is an important obstacle for thetreatment and control of TB. This resistance has traditionally beenattributed to the unusual multi-layer cell envelope and/or activemultidrug efflux pumps.

The acquired drug resistance is mediated by mutations in chromosomalgenes. So far, no single pleiotropic mutation has been found in Mtb tocause a multi-drug resistant (MDR) phenotype. The MDR phenotype iscaused by sequential accumulation of mutations in different genesinvolved in resistance to individual drugs, due to inappropriatetreatment or poor adherence to treatment. However, it is important toobserve that some resistant strains do not present these classicmutations, suggesting the possibility of the existence of othermechanisms such as efflux pumps and alterations in the permeability ofthe cell wall.

Multidrug-resistant TB (MDR-TB) is defined by resistance to the two mostcommonly used drugs in the current four-drug (or first-line) regimen,INH and RMP. According to the World Health Organization (WHO), EasternEurope's rates of MDR-TB are the highest, where MDR-TB makes up 20percent of all new TB cases. During the late 1980s and early 1990s,outbreaks of MDR-TB in North America and Europe killed more than 80% ofthose who contracted the disease. Today, MDR-TB is also quite common inIndia and China, as the two countries combined account for more thanhalf of the global MDR-TB burden.

Drug-resistant TB is the man-made result of interrupted, erratic, orinadequate TB therapy, and its spread is undermining efforts to controlthe global TB epidemic. Multiple drug resistant and extensively drugresistant tuberculosis (XDR-TB) develop when the long, complex,decades-old TB drug regimen is improperly administered, or when peoplewith TB stop taking their medicines before the disease has been fullyeradicated from their body. Once a drug-resistant strain has developed,it can be transmitted directly to others just like drug-susceptible TB.

Treatment for MDR-TB consists of what are called second-line drugs.These drugs are administered when first-line drugs fail. Treatment forMDR-TB is commonly administered for 2 years or longer and involves dailyinjections. Many second-line drugs are toxic and have severe sideeffects. Further, the cost of curing MDR-TB can be staggering—literallythousands of times as expensive as that of regular treatment in someregions—posing a significant challenge to governments, health systems,and other payers.

More recently emerged extensively drug-resistant Mtb strains that arethe agents of extensively drug-resistant tuberculosis (XDR-TB). Thisform of the disease is defined as TB that has developed resistance to atleast RMP and INH, as well as to any member of the quinolone family andat least one of the following second-line anti-TB injectable drugs:kanamycin (KAN), capreomycin (CAP) or amikacin (AMIC).

In recent decades the development of MDR-TB and XDR-TB, and the presenceof HIV have combined to increase the global threat to public healthposed by TB. In addition to increasing individual susceptibility to TBfollowing Mtb infection, a high burden of HIV-associated TB cases alsoexpands Mtb transmission rates at the community level, threatening thehealth and survival of HIV-negative individuals as well. In severalcountries, HIV has been associated with epidemic outbreaks of TB. Manyof the reported outbreaks involved MDR strains, which respond poorly tostandard therapy—the growing burden of TB.

The long and complex regimen is burdensome for patients, even when takenunder direct observation by a healthcare worker or community member, asrecommended by WHO. As a result, many patients do not or cannot completetheir treatment, which leads to the development of drug-resistantstrains. While MDR-TB is a man-made issue, research has shown that thosestrains are now being transmitted from patient to patient. Second-linedrugs are also much more toxic and considerably more expensive than thestandard first-line anti-TB regimen.

Furthermore, current first-line treatment regimens are not compatiblewith certain common antiretroviral (ARV) therapies used to treatHIV/AIDS. To avoid drug-drug interactions in co-infected patients, thetreatment regimen for one of the diseases must be suboptimally modified.Therefore, new drugs are needed that will be effective in treatingchildren, and latent TB infection (an asymptomatic infection) and willbe compatible with antiretroviral therapy. Additionally, new regimensneed to be affordable and easily managed in the field.

The introduction of new drugs, preferably with novel mechanisms ofaction, which will be active against current drug-resistant andextensively drug-resistant strains, and fewer TB drug side effects, willhopefully allow for a shorter TB regimen for both drug-sensitive anddrug-resistant disease (MDR-TB and XDR-TB). Shortening treatment to fouror two months or even less should increase cure rates, improve patientadherence, and lessen the likelihood of developing drug resistance. Thisposes a massive challenge to controlling these twin epidemics, giventhat an estimated one-third of the 40 million people living withHIV/AIDS worldwide are co-infected with TB. The deadly synergy of thesetwo diseases demands first-line treatments that can be fully harmonized.

Drug-resistant TB is difficult, complicated, and expensive to treat.Treatment relies on second-line drugs, and is commonly administered for2 years or longer. It includes daily injections, and often causes severeside effects. Of those who do, nearly half will still die. What's worse,some resistant strains are virtually untreatable with any existingantibiotics. The complexity and prohibitive cost of MDR-TB treatmentmeans that fewer than 3% of the world's MDR-TB patients receive propertreatment. Without a significantly simpler, faster cheaper, oraltreatment for MDR-TB, countries cannot scale up treatment to serve theirpopulations. Without new, simple, and affordable treatments for MDR-TB,this is not realistically possible.

Extensively drug-resistant TB (XDR-TB) is emerging as an even moreominous threat. This makes XDR-TB treatment extremely complicated, ifnot impossible, in resource-limited settings. It is estimated that 70%of XDR-TB patients die within a month of diagnosis. The most recentdrug-resistance surveillance data issued by the WHO estimates that anaverage of roughly 5 percent of MDR-TB cases are XDR-TB.

When drug resistance develops, patients should be treated with a newcombination containing at least three drugs that they had never receivedbefore (or that do not show cross-resistance with those to whichresistance is suspected). In these conditions, the treatment is longer,more toxic, more expensive and less effective than regimens containingfirst-line drugs, and should be directly observed.

Since HIV/AIDS patients have a higher probability of acquiring TB(either pulmonary or extrapulmonary) or other mycobacterialopportunistic infections, particular drug regimens have been designedfor treating active TB disease in them.

Also, the severity of adverse effects of antimycobacterial drugs (due tothe interactions with anti-retroviral drugs) and mortality is higheramong HIV-positive patients. Although, in general, HIV-positive patientsrespond well to a standard short-course treatment of TB, treatmentfailure due to malabsorption of antimycobacterial drugs has beenreported. For example, rifamycins (rifampicin, rifabutin, etc.) haveclinically relevant interactions with some drugs used in theantiretroviral therapy, since they induce the metabolism ofantiretroviral agents such as zidovudine, non-nucleoside reversetranscriptase inhibitors, and HIV protease inhibitors, whoseconcentrations may fall to sub-therapeutic levels. Then, rifamycin-freeregimens have been suggested. They consist of INH, EMB, PZA, andstreptomycin (SM), daily for two months, followed by INH, PZA, and SMtwo or three times weekly for seven months. However, it has also beendescribed that the use of RIF throughout antituberculosis treatmentimproves outcome in HIV patients.

Chemoprophylaxis of TB is indicated for asymptomatic patients having apositive tuberculin skin test (TST) but not showing active disease(latent TB infection), especially when they are at risk of the disease(for example, HIV positive patients). Prophylaxis is most frequentlyachieved by the administration of INH only, at doses of 300 mg daily forsix to nine months (although there is a risk of developing INHresistance). When resistance to INH is suspected, other regimensincluding RIF, PZA or EMB can be administered, although there is agreater chance of having adverse effects. In TB prophylaxis, RIF can begiven concurrently with INH, reducing the prophylaxis treatment to threemonths.

Most drugs used in antituberculosis treatment (isoniazid, rifampicin,rifapentine, rifabutin, pyrazinamide, ethambutol and ethionamide) arecommercially available as tablets or capsules and can therefore be takenorally. Isoniazid is also available as an elixir, in granules forpediatric use, and in aqueous solution for intravenous or intramuscularinjection. Rifampicin is available in powder for preparing suspensionsfor oral administration, and also in aqueous solution for intravenous orintramuscular injection. The exceptions are the aminoglycosides(streptomycin, kanamycin, and amikacin) and capreomycin, which are onlyavailable as aqueous solutions for intravenous or intramuscularinjection. Para aminosalicylic acid (PAS) is usually available asgranules for mixing with food; tablets and solutions for intravenousadministration can also be found. The fluoroquinolones are available astablets or as aqueous solutions for intravenous injections.

Isoniazid, rifampicin and pyrazinamide can also be found in fixed-dosecombination preparations. When available, the use of combinationpreparations is recommended. Indeed, by reducing the number of tabletsto be taken, they facilitate the patient's adherence to treatment andsupervision of therapy. Most importantly, this form of preparationminimizes the possibility of monotherapy and therefore, reduces the riskof drug resistance development.

The framework of mycobacterial infections and diseases all over theworld requires the systematic search for new antimycobacterial drugs.Almost no new antimycobacterial drug classes have been developed overthe last 40 years. In fact, once the industrialized countries feltconfident in accomplishing TB control, the leading pharmaceuticalindustries lost interest in the development of antimycobacterial drugs.

The emergence of the HIV pandemic soon followed by the increase ofMDR-TB and XDR-TB incidence rate, the prevalence of chronic diseases,the generalized use of immunosuppressive treatments, the increase inorgan transplantation and the general increase of the prevalence ofsevere and moderately severe forms of immunodeficiency conditions in thepopulation require an urgent investment in research and development todiscover new candidate compounds to treat the drug-resistant TB,overcome the complex drug-drug interactions between antimycobacterialand antiviral or cytotoxic drugs.

Additionally, these efforts should be focusing in the development of ashorter and simpler regime for TB could improve treatment compliance,stop the spread and enable the global scale-up of MDR-TB and XDR-TBtreatment. A shorter and simpler treatment will not only help cure thosecurrently under care, but will also allow health workers to reach morepeople by reducing the burden on national TB programs.

The research and development of new medicines is an integral part of acomprehensive TB control plan. Without new and improved TB treatmentregimens, including treatment for those suffering from MDR-TB andco-infected with HIV/AIDS, the reduction and eventual eradication of thedisease cannot be achieved.

The nontuberculous mycobacteria (NTM) are for the most part ubiquitousenvironmental organisms found in soil and water that only rarely causedisease in humans. There are numerous species of NTM. Although regionalvariation in species isolation has been shown, the NTM most frequentlyisolated are those of the Mycobacterium avium Complex(MaC)—Mycobacterium intracellulare and Mycobacterium avium,Mycobacterium smegmatis, Mycobacterium kansasii, Mycobacteriumfortuitum, Mycobacterium abscessus and Mycobacterium chelonae.

These organisms have significant structural and biochemical similaritieswith Mtb. Because they are of significantly lower pathogenicity thanMtb, they are considered opportunistic pathogens. NTM are an importantcause of morbidity and mortality, often in the form of progressive lungdisease. Several species are associated with diseases of other organs orsystems (ex.: skin and soft tissue, lymphatic and gastrointestinalsystems). Disseminated disease due to NTM is primarily associated withAIDS and other forms of severe immunosuppression.

Human immunodeficiency virus infection also increases the risk ofdisease mediated by NTM. Patients with AIDS require new treatmentmodalities to approach MaC disease and prevention, such as combinationtherapy with nucleoside reverse transcriptase inhibitors and HIVprotease inhibitors, as well as antimycobacterial prophylaxis. Most ofthe NTM except Mycobacterium kansasii are inherently resistant orpartially susceptible to the standard anti-tubercular drugs.

Drug therapy for MAC disease involves multiple drugs; therefore, therisk of adverse drug reactions and/or toxicities is relatively high. Inaddition, the optimal therapeutic regimen has yet to be established. Therecommended initial regimen for most patients with fibrocavitary ornodular/bronchiectatic MaC lung disease is a three times weekly regimenincluding clarithromycin or azithromycin, ethambutol and rifampinadministered three times per week. Patients respond best to MaCtreatment regimens the first time they are administered; therefore, itis very important that patients receive recommended multidrug therapythe first time they are treated.

The management of macrolide-resistant MaC involves complex clinicaldecision making, drug choices, and protracted duration of therapy,analogous to the drug management of MDR-TB.

Multiple factors can interfere with the successful treatment of MaC lungdisease, including medication nonadherence, adverse events, priortherapy of MaC lung disease, lack of response to a medication regimen,or the emergence of a macrolide-resistant MaC strains.

The Mycobacterium leprae (Mlp) is the etiologic agent of leprosy, achronic mycobacterial disease characterized by the involvement primarilyof skin as well as peripheral nerves and the mucosa of the upper airway.The organism has never been grown in bacteriologic media or cellculture, but has been grown in mouse foot pads. The risk groups are theclose contacts with patients with untreated, active, predominantlymultibacillary disease and persons living in countries with highlyendemic disease.

In 2002, Brazil, Madagascar, Mozambique, Tanzania, and Nepal had having90% of cases. Worldwide, 1-2 million persons are permanently disabled asa result of leprosy. However, persons receiving antibiotic treatment orhaving completed treatment are considered free of active infection.

Multidrug therapy has not been implemented in many endemic areas. Nervedamage must be recognized and managed. Relapse rate after completion ofshort course multidrug therapy may rise.

Paucibacillary leprosy should be treated for 6-12 months with dapsoneplus rifampin. This regimen should be followed by treatment with dapsoneas monotherapy for 3 years in patients with tuberculoid leprosy or 5years in patients with borderline lepromatous leprosy. Multibacillaryleprosy should be treated for months with dapsone 100 mg/day,clofazimine 50 mg/day and rifampin 600 mg plus clofazimine 300 mg/month.Increasing resistance in patients treated for leprosy has been reportedin Southeast Asia. The drug most commonly found to be resistant isdapsone, often in the context of prior exposure or treatment attemptswith monotherapy.

More effective and safer compounds should allow simpler and shortermultiple-drug regimes, with a reduced risk of interactions with HIV/AIDSdrug treatment or with immunosuppressive and cytotoxic drugs. Thedevelopment of drug-resistant strains is a continuous biologicalprocess, accelerated by the noncompliance with the available regimes andthe increase of moderately severe and severe forms of imunodepression inlow- and high-income countries. The treatment and prevention of diseasescaused by microorganisms of the Mycobacterium genus (e.g., Mycobacteriumtuberculosis, nontuberculous mycobacteria and Mycobacterium leprae)requires, urgently, an investment in the research and development of newcompounds.

Therefore, the technical problem solved in the present invention is toprovide further pharmaceutical active compounds for the prevention andtreatment of tuberculosis as well as diseases caused by nontuberculousmycobacteria or caused by Mycobacterium leprae. Surprisingly, theinventors have found that the compounds of formula I are effective inthe prevention and treatment of tuberculosis, diseases caused bynontuberculous mycobacteria and/or caused by Mycobacterium leprae.

SUMMARY OF THE INVENTION

The invention provides pyridyl-hydrazones of Formula I, orpharmaceutically acceptable salts, esters, solvates, isomers andprodrugs thereof as well as pharmaceutical compositions containing thesecompounds for use in the prophylactic and/or therapeutic treatment oftuberculosis and related diseases, such as, diseases caused bynontuberculous mycobacteria and/or caused by Micobacterium leprae.

Compounds of the present invention are represented by the followingFormula I:

where Q is

andA is a 5-6 member substituted or unsubstituted aryl or heteroaryl ringselected from:

whereinR¹, R² and R³ are each independently of one another, hydrogen,(C₁-C₄)alkyl, hydroxyl, halogen, methoxy or methoxy-acetic acid;andR⁴ and R⁵ are each independently of one another, hydrogen or(C₁-C₄)alkyl,and pharmaceutical acceptable salts, esters, solvates, isomers andprodrugs thereof.

Preferred compounds of the general Formula I are represented by thefollowing Formula Ia, and Formula Ib:

Formula (Ia) compounds are represented by:

wherein R¹, R² and R³ are each independently of one another, hydrogen,(C₁-C₄)alkyl, hydroxyl, halogen, methoxy or methoxy-acetic acid;and pharmaceutical acceptable salts, esters, solvates, isomers andprodrugs thereof.

And Formula (Ib) compounds are represented by:

wherein R⁴ and R⁵ are each independently of one another, hydrogen or(C₁-C₄)alkyl,and pharmaceutical acceptable salts, esters, solvates, isomers andprodrugs thereof.

Most preferred compounds of the present invention are selected from thegroup consisting of:

-   -   N-pyridin-2-yl-N′-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine,    -   N-pyridin-2-yl-N′-5-methyl-2-hydroxyphenylmethylene-hydrazine,    -   N-pyridin-2-yl-N′-5-bromo-2-hydroxyphenylmethylene-hydrazine,        and    -   N-pyridin-2-yl-N′-thiophen-2-ylmethylene-hydrazine.

Further embodiments of the present invention contemplatepharmaceutically acceptable salts, esters, solvates, isomers andprodrugs of compounds of Formula I and pharmaceutical compositionscomprising a compound of Formula I, or a pharmaceutically acceptablesalt, ester, solvate, isomer or prodrug thereof, in combination with apharmaceutically acceptable diluent or carrier for use in the treatmentand/or prevention of tuberculosis and related diseases, such as diseasescaused by nontuberculous mycobacteria and/or caused by Micobacteriumleprae.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates Mycobacterium smegmatis growth inhibition results forcompounds tested.

FIG. 2 illustrates Mycobacterium smegmatis growth inhibition results forpreferred compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention, there is provided a compoundrepresented by the following Formula I:

where Q is

and A is a 5-6 member substituted or unsubstituted aryl or heteroarylring selected from:

whereinR¹, R² and R³ are each independently of one another, hydrogen,(C₁-C₄)alkyl, hydroxyl, halogen, methoxy or methoxy-acetic acid;andR⁴ and R⁵ are each independently of one another, hydrogen or(C₁-C₄)alkyl,and pharmaceutical acceptable salts, esters, solvates, isomers andprodrugs thereof.

In the context of the present invention the term “(C₁-C₄)alkyl” meansmethyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl or tert-butyl,and the term “halogen” means fluorine, chlorine, bromine or iodine.

In a further aspect of the present invention, there is providedcompounds falling under the definition of the general Formula I andrepresented by the following Formula Ia and Formula Ib:

Formula (Ia) compounds are represented by:

-   -   wherein R¹, R² and R³ have the same meaning as defined above.

And, Formula (Ib) compounds are represented by:

wherein R⁴ and R⁵ have the same meaning as defined above.

In yet another aspect of the present invention, there are providedcompounds of Formula I selected from the group consisting of:N-pyridin-2-yl-N′-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine,N-pyridin-2-yl-N′-5-methyl-2-hydroxyphenylmethylene-hydrazine,N-pyridin-2-yl-N′-5-bromo-2-hydroxyphenylmethylene-hydrazine andN-pyridin-2-yl-N′-thiophen-2-ylmethylene-hydrazine.

In a further aspect of the present invention, there is provided acompound of Formula I, Ia and/or Ib, or a pharmaceutical acceptablesalt, ester, solvate, isomer or prodrug thereof, useful in the treatmentof a disease in a mammal, such as a human.

In yet another aspect of the present invention there is provided apharmaceutical composition comprising: (a) a therapeutically effectiveamount of a compound of Formula I, Ia, and/or Ib; or a pharmaceuticalacceptable salt, ester, solvate, isomer or prodrug thereof, and (b) apharmaceutically acceptable excipient useful for the treatment of adisease in a mammal, such as human.

In a further aspect of the present invention, there is provided acompound of Formula I, Ia and/or Ib, or a pharmaceutical acceptablesalt, ester, solvate, isomer or prodrug thereof, or a pharmaceuticalcomposition comprising said compound for inhibiting pantothenatesynthetase enzyme activity.

In a yet a further aspect of the present invention, there is provided acompound of Formula I, Ia and/or Ib, or a pharmaceutical acceptablesalt, ester, solvate, isomer or prodrug thereof or a pharmaceuticalcomposition comprising said compound for preventing and/or treatingtuberculosis and related diseases, such as diseases caused bynontuberculous mycobacteria and/or caused by Micobacterium leprae.

The present invention is intended to encompass all pharmaceuticallyacceptable ionized forms (e.g., salts) and solvates (e.g., hydrates) ofthe compounds of Formula I, Ia and Ib, regardless of whether such formsand solvates are specified, as it is well known in the art thatpharmaceutical agents in an ionized or solvated form may be used.

Compounds of Formula I, Ia and Ib may contain one or more chiral centersand exist in optically active forms. When a compound of Formula I, Iaand/or Ib or a salt thereof contains a single chiral center, it mayexist in two enantiomeric forms. The present invention includesindividual enantiomers and mixtures of these enantiomers, which may beobtained by methods known to those skilled in the art.

When a compound of Formula I, Ia and Ib or a salt thereof contains morethan one chiral center it may exist in diastereomeric forms. The presentinvention includes each diastereomer and mixtures of thesediastereomers, which may be obtained by methods known to those skilledin the art.

The compounds under Formula I, Ia and Ib may form organic and inorganicsalts, for example, with inorganic or organic acids, e.g., hydrochloricacid, hydrobromic acid, fumaric acid, tartaric acid, citric acid,sulfuric acid, maleic acid, acetic acid, succinic acid, benzoic acid,palmitic acid, dodecanoic acid and acidic amino acids, such as glutamicacid, alkali metal hydroxides, e.g., sodium hydroxide, with amino acids,e.g., lysine or arginine. The salts formed with compounds under FormulaI, Ia and Ib, provided that they are pharmaceutically acceptable may beused in the present invention. Such salts and corresponding solvatesalso fall within the scope of the present invention.

Prodrugs of the compounds under Formula I, Ia and Ib are also thesubject of the present invention. As is known in the art, prodrugs arealtered in vivo and become a compound of the present invention. Allstandard methods of using the compounds of the present invention areintended, whether prodrug delivery is specified, to encompass theadministration of a prodrug that is converted in vivo to a compoundaccording to the present invention.

A variety of routes of administration of the compounds and compositionsof the present invention are possible including, but not necessarilylimited to parenteral (e.g., intravenous, intra-arterial, intramuscular,subcutaneous injection), oral (e.g., dietary or by inhalation), topical,nasal, rectal, or via slow release micro-carriers, depending on thedisease or condition to be treated. Oral, parenteral and intravenousadministrations are preferred modes of administration. The formulationof the compounds of the present invention to be administered will varyaccording to the route of administration selected (e.g., solution,emulsion, gel, aerosol, capsule). Further dosage forms according to thepresent invention are, for example, solutions, suspensions, ointments,creams, pastes, gels, tinctures, lip-sticks, drops, syrups, aerosols andsprays.

An appropriate composition of the present invention comprising thecompound or compounds of Formula I, Ia and/or Ib can be prepared in aphysiologically acceptable vehicle or carrier and optional adjuvant andpreservatives. For solutions or emulsions, suitable carriers include,for example, aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media, sterile water, creams,ointments, lotions, oils, pastes and solid carriers. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. Intravenous vehiclescan include various additives, preservatives, or fluid, nutrient orelectrolyte replenishers. (See Remington's Pharmaceutical Science,16^(th) Edition, Mack, Ed. (1980))

The preferred compositions for parenteral administration are under theform of solutions, suspensions, emulsions, dispersions and lyophilizedcompositions of the compounds of the invention, preferably in the formof isotonic aqueous solutions, dispersions, emulsions or suspensions.These compositions are preferably sterile, either being processed in asterile environment during their whole preparation process or by beingsterilized in the end of said process. Furthermore, their manufacture isusually carried out under sterile conditions, as is the filling, forexample, into ampoules or vials, and the sealing of the containers.These compositions may be ready to apply or be presented under solidform (for example as a lyophilizate) requiring reconstitution priorapplication.

Parenteral compositions according to the present invention may compriseexcipients, for example vehicles, stabilizers (reducing agents,anti-oxidants and/or sequestering agents), buffering agents,preservatives, isotonizing agents, emulsifiers, solubilizers, viscosityincreasing agents, and/or bulking agents and are prepared byconventional processes well known to those knowledgeable of the art.

Non-limiting examples of vehicles, in the context of the presentinvention, include water for injections, oily vehicles, polyethyleneglycol, benzyl alcohol, ethanol and glycerol.

Non-limiting examples of oily vehicles in the context of the presentinvention include fatty acid esters and mixtures of fatty acid esters,vegetable oils, synthetic oils and semisynthetic oils, almond oil,castor oil, cottonseed oil, groundnut oil, olive oil, sesame oil, andsoybean oil.

Non-limiting examples of reducing agents in the context of the presentinvention include sodium sulfite, sodium bisulfite and sodiummetabisulfite.

Non-limiting examples of anti-oxidants in the context of the presentinvention include butylated hydroxyanisole, gallic acid esters andtocopherols.

Non-limiting examples of sequestering agents in the context of thepresent invention include ethylenediaminetetraacetic acid in the form ofsodium salt (EDTA), tartaric acid, thiourea and monothioglycerol.

Non-limiting examples of buffering agents in the context of the presentinvention include the combination of monosodium phosphate with disodiumitself, trisodium phosphate, urea, sodium borate and sodium citrate.

Non-limiting examples of preservatives in the context of the presentinvention include methylparaben, the cresols, benzyl alcohol andphenyllic alcohol.

Non-limiting examples of isotonizing agents in the context of thepresent invention include boric acid, calcium gluconate, chlorobutanol,potassium chloride, sodium citrate, sodium borate, sodium phosphate,sodium chloride and sodium lactate.

Non-limiting examples of emulsifiers in the context of the presentinvention include lecithins, monoglycerides, polyethylene polymers andpolypropylene polymers.

Non-limiting examples of solubilizers in the context of the presentinvention include ethanol, polypropylene glycol, N,N-dimethylacetamideor polyoxyethylene sorbitan esters.

Non-limiting examples of viscosity-increasing agents in the context ofthe present invention include sodium carboxymethylcellulose,carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatins.

Non-limiting examples of bulking agents in the context of the presentinvention include mannitol, lactose, sucrose, trehalose, sorbitol,glucose, raffinose, arginine, glycine, histidine, dextran orpolyethylene glycol.

Pharmaceutical oral compositions in the solid oral form (tablets, softcapsules, hard capsules or any other) according to the present inventioncomprise excipients, provided they are compatible with the activeingredient of the composition, including, but not limited to, diluents,binders, disintegrants, surfactants, glidants, lubricants, antioxidantsor free radicals sequestrants, coating components, opacifiers orplasticizers.

Soft capsules in the context of the present invention consist of gelatinor any other suitable substance containing the compounds of theinvention dissolved, emulsified or suspended in a suitable soft capsulevehicle and optionally excipients such as stabilizers (reducing agents,anti-oxidants and/or sequestering agents, as defined above),solubilizers (as defined above), plasticizers or others. Hard capsulesin the context of the present invention may in addition to the compoundsof the invention also optionally contain excipients such as fillers,glidantes or others.

Non-limiting examples of diluents in the context of the presentinvention include cellulose preparations, calcium phosphates, anhydrouslactose, monohydrate lactose, dihydrate lactose, sorbitol, starch,pregelatinized starch, sucrose and mannitol.

Non-limiting examples of binders in the context of the present inventioninclude sodium carboxymethyl cellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, methylcellulose, hydroxypropylcellulose,povidone, a starch paste, pregelatinized starch and sucrose.

Non-limiting examples of disintegrants in the context of the presentinvention include sodium carboxymethyl cellulose, microcrystallinecellulose, croscarmellose sodium, crospovidone, hydroxypropylcellulose,povidone, poloxamer, sodium lauryl sulfate, starch, pregelatinizedstarch sodium glycolate, alginic acid or a salt thereof, such as sodiumalginate.

Non-limiting examples of surfactants in the context of the presentinvention include poloxamer and sodium lauryl sulfate.

Non-limiting examples of glidants in the context of the presentinvention include calcium silicate, starch, talc, colloidal silicondioxide, magnesium stearate and sodium aluminum silicate.

Non-limiting examples of lubricants in the context of the presentinvention include sodium stearyl fumarate, sodium lauryl sulfate, talc,silicic acid, stearic acid or salts thereof, such as magnesium orcalcium stearate, and/or polyethylene glycol, or derivatives thereof.

Non-limiting examples of antioxidants and free radical scavengers in thecontext of the present invention include butilhidroxiltoluen,butilhidroxilanisol, citric acid and citrate salts, ascorbate salts andascorbate, alpha-tocopherol, sodium acetate, sodium sulphite andcompound with organic thiol function.

Non-limiting examples of coating components in the context of thepresent invention include concentrated sugar solutions, gum arabic,talc, polyvinylpyrrolidone, polyethylene glycol, titanium dioxide,coating solutions in suitable organic or mixed solvents, cellulosephtalates (acetylcellulose phthalate or hydroxypropylmethylcellulosephthalate).

Non-limiting examples of plasticizers in the context of the presentinvention include glycerol and sorbitol.

Non-limiting examples of soft capsule vehicles in the context of thepresent invention include fatty oils, paraffin oil, liquid polyethyleneglycols or ethylene/propylene glycol fatty acid esters.

Suppositories according to the present invention comprise a compound ofthe present invention admixtured with a suppository base and optionallyfurther excipients.

Non-limiting examples of suppository bases in the context of the presentinvention include natural or synthetic triglycerides, paraffinhydrocarbons, polyethylene glycols or higher alkanols (alkanols with atleast eight carbon atoms).

The term “pharmaceutically acceptable carrier” as used in the presentinvention includes any solvent, dispersion media, coatings,antibacterial, and antifungal agents, isotonic and absorption delayingagents, and the like which are compatible with the activity of thecompounds and are physiologically acceptable to the subject.

“Effective amount” a used in the present invention includes the amountof the compound or a pharmaceutically acceptable salt thereof, ester,isomer, solvate, or prodrug thereof which allows it to perform itsintended function, i.e., prevention of onset or treatment oftuberculosis or related diseases such as caused by nontuberculousmycobacteria and/or caused by Mycobacterium leprae.

A therapeutically effective amount of the active substance of thepresent invention can be administered by an appropriate route in asingle dose or multiple doses.

The therapeutically effective amount will depend upon a number offactors, including biological activity, mode of administration,frequency of treatment, type of concurrent treatment, if any, age, bodyweight, sex, general health, severity of the condition to be treated, aswell as appropriate pharmacokinetic properties. One skilled in the artcan determine the appropriate dosage based on the above factors.

The compounds of the invention may be administered initially in asuitable dosage that may be adjusted as required, depending on theclinical response. In general, satisfactory results may be obtained whenthe compounds of the invention are administered to a human at a dailydosage of between 0.01 mg and 5000 mg (measured as the solid form). Apreferred dose ranges between 0.01 and 750 mg/Kg, more preferablybetween 0.05 and 150 mg/Kg.

The compound can be administered in the form of pharmaceuticalcompositions comprising the compound once a day or at different timeswithin the day, prophylactically or therapeutically, preferably in anamount effective against tuberculosis or the related disease, to amammal, for example a human, requiring such treatment. In the case of anindividual having a bodyweight of about 70 kg, the daily dose of themixture administered is from approximately 0.01 g to approximately 50 g,preferably from approximately 0.05 g to approximately 10 g, of acompound of Formula I, Ia and/or Ib.

The pharmaceutical compositions of the present invention comprise fromapproximately 5% to approximately 95% of a mixture of a compound offormula I.

The pharmaceutical compositions of the present invention may, ifdesired, be formulated so as to provide an immediate or modified releaseof the active ingredient after administration to the patient.

Unit dose administration forms according to the present inventioncomprise from approximately 20% to approximately 90% of the compound offormula I, and forms that are non-unit dose type from approximately 5%to approximately 20% of the mentioned compound. Unit dose formsaccording to the present invention refer to, for example, coated anduncoated tablets, microcapsules, soft and hard capsules, pellets,powdered doses, ampoules, vials and suppositories.

The present invention relates especially to the use of a compound offormula I, Ia and/or Ib or a pharmaceutical acceptable salt, ester,isomer, solvate and/or prodrug, as such or in the form of apharmaceutical formulation with at least one pharmaceutically acceptablecarrier for the therapeutic and also prophylactic treatment oftuberculosis.

The pharmaceutical compositions of the invention are not only useful forthe prevention and treatment of tuberculosis, i.e. a disease caused byMycobacterium tuberculosis complex (MTbC), but also for the treatment ofdiseases caused by related bacteria, in particular Mycobacterium leprae,and caused by nontuberculous mycobacteria (NTM).

Mycobacterial diseases are caused by organisms of the Mycobacteriumtuberculosis complex (MtbC) like Mycobacterium tuberculosis (Mtb),Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetii andMycobacterium microti. Mycobacteria other than MtbC and Mycobacteriumleprae are known as non-tuberculous mycobacteria (NTM) and can causealso human and animal diseases as is the case for Mycobacterium aviumcomplex (MAC), Mycobacterium smegmatis, Mycobacterium gordonae,Mycobacterium kansasii, Mycobacterium terrae, Mycobacteriumscrofulaceum, Mycobacterium vaccae, Mycobacterium marinum, Mycobacteriumlentiflavum, Mycobacterium fortuitum, Mycobacterium chelonae,Mycobacterium abscessus, Mycobacterium intracellulare and Mycobacteriumavium.

As above mentioned, multidrug-resistant TB (MDR-TB) is defined byresistance to the two most commonly used drugs in the current four-drug(or first-line) regimen, INH and RMP. Drug-resistant TB is the man-maderesult of interrupted, erratic, or inadequate TB therapy, and its spreadis undermining efforts to control the global TB epidemic.Multidrug-resistant (MDR-TB) and extensively drug resistant tuberculosis(XDR-TB) develop when the long, complex, decades-old TB drug regimen isimproperly administered, or when people with TB stop taking theirmedicines before the disease has been fully eradicated from their body.Extensively drug resistant tuberculosis (XDR-TB) is defined as TB thatis resistant to any fluoroquinolone, and at least one of threeinjectable second-line drugs (capreomycin, kanamycin, and amikacin), inaddition to INH and RMP.

The pharmaceutical compositions of the invention are effective in thetreatment and prevention of all forms of tuberculosis such as primarytuberculosis disease, post-primary pleuropulmonary tuberculosis disease,post-primary extra-pulmonary tuberculosis disease involving at least oneorgan or system such as, but not restricted, to lymph nodes, kidney,central nervous system, osteoarticular systems, gastrointestinal systemtract, eye, skin and soft tissues or urogenital system, disseminatedtuberculosis and reactivated tuberculosis.

The pharmaceutical compositions of the invention are effective in thetreatment and prevention of all forms of tuberculosis in adults,children and elderly patients, with or without immunodepressionconditions such as, but not restricted to, diabetes mellitus, chronicrenal insufficiency, malnutrition, alcoholism, human immunodeficiencyvirus infection (HIV)/acquired immunodeficiency syndrome (AIDS),silicosis, paracoccidioidomycosis, leukemias, solid tumors,immunosuppressive drug treatments and hereditary diseases or syndromes.

The pharmaceutical compositions of the invention are effective in thetreatment and prevention of diseases caused by non-tuberculousmycobacteria that included at least one organ or system such as, but notrestricted to, lungs and endobronchial tree, lymph nodes, kidney,central nervous system, osteoarticular system, gastrointestinal system,eye, skin and soft tissues, urogenital system, disseminated andreactivated forms of disease.

The pharmaceutical compositions of the invention are effective in thetreatment and prevention of diseases caused by nontuberculousmycobacteria in adults, children and elderly patients, with or withoutimmunosuppressive conditions such as, but not restricted to, diabetesmellitus, chronic renal insufficiency, malnutrition, alcoholism, humanimmunodeficiency virus infection (HIV)/acquired immunodeficiencysyndrome (AIDS), leukemias, solid tumors, immunosuppressive drugtreatments and hereditary diseases or syndromes.

The pharmaceutical compositions of the invention are effective in thetreatment and prevention of diseases caused by Mycobacterium leprae thatincluded at least one organ or system such as, but not restricted to,skin and soft tissues, urogenital system and central nervous system,disseminated and reactivated forms of disease.

The pharmaceutical compositions of the invention are effective in thetreatment and prevention of diseases caused by Mycobacterium leprae inadults, children and elderly patients, with or without immunosuppressiveconditions such as, but not restricted to, diabetes mellitus, chronicrenal insufficiency, malnutrition, alcoholism, human immunodeficiencyvirus infection (HIV)/acquired immunodeficiency syndrome (AIDS),leukemias, solid tumors, immunosuppressive drug treatments andhereditary diseases or syndromes.

Obtaining the Compounds of the Present Invention

All compounds are available at Chembridge Corporation(www.chembridge.com), however the skilled technician can easily obtainthem by applying various synthetic methods described in the literature:

Method 1) for Obtaining Compounds of Formula I, in Particular, Compoundsof Formula (Ia)

Reaction of 2-chloropyridine with hydrazine hydrate at a refluxtemperature results in the formation of 2-hydrazinopyridine. Furthercondensation with different aldehydes (for instance, in ethanol at areflux temperature) gives the corresponding hydrazone. The synthesismethod is described in Chaur, Manuel N. et al.; Chemistry—A EuropeanJournal; vol. 17; nb. 1; (2011); p. 248-258; Padalkar, Vikas S. Et al.;Synthetic Communications; vol. 41; nb.6; (2011); p. 925-938; Fargher;Furness; Journal of the Chemical Society; vol. 107; (1915); p.695

Method 2) for Obtaining Compounds of Formula I, in Particular Compoundsof Formula (Ib)

These compounds can be obtained with good overall yield from2-aminopyridine which is transformed in the corresponding 2-bromoderivative by treatment with bromine and bromidric acid. The 2-bromocompound obtained is then treated in SNAr conditions with hydrazinehydrate to provide the desired 2-hydrazinepyridine derivative. Finally,the compounds are prepared by condensation of the 2-hydrazinepyridinederivative (in acidic ethanolic solution) at room temperature with theappropriated heteroarylaldehyde, producing the desired compounds in goodyield. The synthesis method is described in Todeschini, Adriane R. etal.; European Journal of Medicinal Chemistry; vol. 33; nb.3; (1998); p.189-200.

Obtaining the Pharmaceutical Compositions of the Present Invention

The pharmaceutical compositions of the present invention are prepared ina manner known per se, for example by means of conventional mixing,granulating, coating, dissolving, emulsifying or lyophilizing processes.Optionally, the manufacture of the compositions according to the presentinvention includes more steps such as liposomal encapsulation.

In particular, a tablet may be made by compression and molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the active compoundof the present invention in a free-flowing form, e.g., a powder orgranules, optionally mixed with ingredients, such as, binders,lubricants, inert diluents, surface active or dispersing agents. Moldedtablets may be made by molding in a suitable machine, a mixture of thepowdered active compound with any suitable carrier.

In particular, a syrup or suspension may be made by adding the activecompound of the present invention to a concentrated, aqueous solution ofa sugar, e.g., sucrose, to which also any accessory ingredient may beadded. Such accessory ingredients may include, flavoring, an agent toretard crystallization of the sugar or an agent to increase thesolubility of any other ingredient, e.g., as a polyhydric alcohol, forexample, glycerol or sorbitol.

Formulations for rectal administration may be made with a conventionalcarrier, e.g., cocoa butter or Witepsol S55 (commercial registeredtrademark). Specific details related to particular aspects ofconventional processes of galenic development can be found in Swarbrickand Boylan's “Encyclopedia of pharmaceutical technology” (1988-2001 NY,Published by M. Dekker).

Alternatively, the compounds of the present invention may be made inliposomes or microspheres (or microparticles), such methods essentiallycomprising dissolving the compounds of the present invention in anaqueous solution, the appropriate phospholipids and lipids added, alongwith surfactants if required, and the material dialyzed or sonicated, asnecessary. Liposomal encapsulation techniques detailed in ClaudioNastruzzi's book “Lipospheres in drug targets and delivery: approaches,methods, and applications” (Boca Raton 2005, published by CRC Press) andin Lasic and Papahadjopoulos' “Lipospheres in drug targets and delivery:approaches, methods, and applications” (1998 Amsterdam, N.Y., Publishedby Elsevier).

Development and Validation of a High-Throughput Screening (HTS) Platform

Regarding the experimental phase, the present invention included thedevelopment and validation of a high-throughput screening (HTS) platformthat involves the targeting of pantothenate synthetase, an enzymeessential to the metabolic pathways of Mycobacterium tuberculosis, byusing small molecules as potential inhibitors of this enzyme.

The HTS platform was developed, validated and used to screen 50.000compounds that were obtained from a diverse chemical library supplied bya vendor called Chembridge.

The HTS platform consists in a yeast strain genetically modified toexpress the Mycobacterium tuberculosis's pantothenate synthetase gene(PanC) instead of the regular Pan6 of the wild-type yeast. Additionally,this PanC yeast strain is completely dependent on the activity of theexpressed pantothenate synthetase to survive.

The validation of the platform demonstrated that (in normal conditions):

-   -   Wild-type yeast (Control)—shows normal growth;    -   Yeast without Pan6—shows reduced growth;    -   Yeast without Pan6 and complemented with PanC—shows normal        growth (similar to control);    -   Yeast without Pan6 and complemented with Pan6—shows normal        growth (similar to control which proves that the genetic        intervention does not compromise the metabolic processes of the        yeast).

Therefore, a potential inhibitor of the pantothenate synthetase enzymewould result in a reduced growth of the PanC yeast strain.

At the end of this phase the obtained results allowed the classificationof a ranking of compounds that included the most potent in inhibitingthe growth of the yeast that carried the target, pantothenate synthetaseof Mycobacterium tuberculosis.

Based on the data obtained in the screening assay, a ranking ofcompounds was established and the top compounds were selected based on acurve fitting approach that allowed the determination of inhibitoryconcentrations (IC₅₀ and IC₉₀) for each screened compound.

For each compound, a concentration-response curve was created using afour parameter logistic fit (available in the curve-fitting softwareIBDS XLfit™ curve fitting software) that allowed the calculation of 10₅₀and 10₉₀ values for each compound.

Additionally, an in vitro assay using Mycobacterium smegmatis (as asurrogate for anti-Mycobacterium tuberculosis activity) was developed,implemented and used to screen the compounds of interest in anexperimental assay. It is important to mention that Mycobacteriumsmegmatis is considered to be a valuable tool for screening of compoundspotentially active against Mycobacterium tuberculosis, not only becauseof its non-pathogenic profile but also because of the known genehomology between the two mycobacteria species (Andries K et al., Adiarylquinoline drug active on the ATP synthase of Mycobacteriumtuberculosis. Science. 2005; 307:223-227; Chatuverdi V et al.,Evaluation of Mycobacterium smegmatis as a possible surrogate screen forselecting molecules active against multi-drug resistant Mycobacteriumtuberculosis. J Gen Appl Microbiol. 2007; 53:333-337; Cho Y, Ioerger T Rand Sacchettini. Discovery of Novel Nitrobenzothiazole Inhibitors forMycobacterium tuberculosis ATP phosphoribosyl Transferase (HisG) throughvirtual screening. J Med Chem. 2008; 51:5984-5992).

The experimental assay with Mycobacterium smegmatis involves theincubation of the test items and the control items with Mycobacteriumsmegmatis in 5 concentrations (1.24, 3.7, 11, 33 and 100 μM) for 96hours. In terms of control items, a blank control that will exhibitmaximum growth and a positive control that will exhibit inhibitedcontrol are used in this assay. The microorganism growth is evaluated bythe measurement of the suspension turbidity: the comparison betweennegative control and compound or positive control turbidity allowed forthe evaluation of the growth inhibition due to the compound presence.The positive control compound used was rifampicin (RMP) that wasdetermined to be the most effective against this strain of Mycobacteriumsmegmatis. Additionally, it is important to notice that RMP is afirst-line treatment drug used against Mycobacterium tuberculosis in theclinical setting.

After obtaining the concentration-response data for each of thecompounds tested in this Mycobacterium smegmatis screening assay, acurve fitting approach based on a four parameter curve fit, allowed forthe determination of inhibitory concentrations values such as the IC₅₀that represents the required concentration to inhibit 50% of theMycobacterium smegmatis growth. This assay had the objective ofconfirming the superior inhibitory activity of the compounds of thepresent invention over those known from the prior art, such as RMP.

The results obtained through these assays demonstrate that the followingcompound: 3,5-dibromo-2hydroxybenaldehyde (2-pyridinyl)hydrazine,denominated compound 42, show a superior inhibitory activity againstMycobacterium smegmatis than the prior art compound RMP.

Further and in order to define analogues of the compoundN-pyridin-2-yl-N′-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine(compound 42) with the desired pharmacological activity, fingerprint andpharmacophore searching methods were carried out using browser-basedsearch engines. In the case of the fingerprint searching method, 2Dsubstructural fragments ofN-pyridin-2-yl-N′-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine(compound 42) were used to search online databases for other moleculesthat share the same 2D substructures and therefore calculate thesimilarity of both molecules as a function of the number of fragmentsthat they have in common. For the pharmacophore search, the compoundN-pyridin-2-yl-N′-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine(compound 42) was used as the pharmacophore model, including thecorrespondent pharmacophoric pattern that involves its ensemble ofsteric and electrostatic features, and online databases withpre-computed conformations were searched for analogues. For bothmethodologies, only the analogues with a similarity higher than 90% werechosen and tested in the same Mycobacterium smegmatis assay describedabove.

The results obtained from this assay locate additional compounds fallingunder the formula I, Ia and Ib bearing the desired pharmacologicalactivity. These compounds are in particular:N-pyridin-2-yl-N′-5-methyl-2-hydroxyphenylmethylene-hydrazine,N-pyridin-2-yl-N′-5-bromo-2-hydroxyphenylmethylene-hydrazine andN-pyridin-2-yl-N′-thiophen-2-ylmethylene-hydrazine.

EXAMPLES

The invention will now be further described by the following workingexamples, which are preferred embodiments of the invention. Theseexamples are illustrative rather than limiting and it is to beunderstood that there may be other embodiments that fall within thespirit and scope of the invention as defined by the claims appendedhereto.

Example 1 Identification of Compounds with Pantothenate SynthetaseInhibiting Activity

The screening step in the HTS platform consisted in the incubation ofthe library compounds (50.000 diverse and publically available chemicalentities) with the yeast complemented with PanC and the comparison ofits growth at the end of 72 hours with the growth observed in thecontrol yeast. This was performed in a high-throughput manner usingrobotized procedures and 96-wells plaques for the compound and yeastincubation. The screening step involved the use of 5 concentrations ofeach compound (0.5, 1, 5, 10 and 20 μM). The comparison of the growthobserved for the control yeast with the growth observed for eachscreened compound allowed the determination of growth inhibitionpercentage for each tested concentration.

At the end of this phase the obtained results allowed the classificationof a ranking of compounds that included the most potent in inhibitingthe growth of the yeast that carried the target, pantothenate synthetaseof Mycobacterium tuberculosis.

From this ranking of compounds, the top compounds were selected based ona curve fitting approach that allowed the determination of inhibitoryconcentrations (IC₅₀ and IC₉₀) for each screened compound.

For each compound, a concentration-response curve was created using afour parameter logistic fit (available in the curve-fitting softwareIBDS XLfit™ curve fitting software) that allowed the calculation of IC₅₀and IC₉₀ values for each compound. The fit quality of each curve wasdetermined by the calculation of curve fit quality values (r²).

Example 2 In Vitro Assay with Mycobacterium smegmatis for ScreeningCompounds with Anti Mycobacterium tuberculosis Activity

Each of the compounds tested and the positive control were incubatedwith Mycobacterium smegmatis in 5 concentrations (100, 33, 11, 3.7 and1.24 μM) for 96 hours. Mycobacterium smegmatis incubation was performedin suspension mode, without agitation, in Middlebrook 7H9 mediasupplemented with ADC, at 37° C. The microorganism growth was evaluatedby the measurement of the suspension turbidity: the comparison betweennegative control and compound or positive control turbidity allowed forthe evaluation of the growth inhibition due to the compound presence.The positive control compound used was RMP that was determined to be themost effective against this strain of Mycobacterium smegmatis.Additionally, it is important to notice that RMP is a first-linetreatment drug used against Mycobacterium tuberculosis in the clinicalsetting.

Based on the results, which are shown in FIGS. 1 and 2, we can observethat there was one compound,N-pyridin-2-yl-N′-3,5-dibromo-2-hydroxyphenylmethylene-hydrazine,denominated compound 42, that exhibited an inhibitory profile ofMycobacterium smegmatis growth similar to the one showed by the activecontrol, RMP.

Example 3 Obtaining Analogues of Most Effective Compound

A series of analogues of the compound,3,5-dibromo-2-hydroxybenzaldehyde(2-pyridinyl)hydrazine (compound 42)were obtained through fingerprint and pharmacophore searching methodsand only the analogues with a similarity higher than 90% were chosen andtested in the same Mycobacterium smegmatis assay described above. Thefollowing table comprise some examples of compounds representative ofthe whole serie:

TABLE 1 Example of Compounds of Formula Ia

Compound no R¹ R² R³ 42 -(2)-OH -(3)-Br -(5)-Br 61 -(2)-OH H -(5)-CH₃ 70-(2)-OH H -(5)-Br Compound no. 42: 3,5-dibromo-2-hydroxybenzaldehyde(2-pyridinyl)hydrazine Compound no. 61:2-hydroxy-5-methylbenzaldehyde-(2-pyridinyl)hydrazine. Compound no. 70:5-bromo-2-hydroxybenzaldehyde-2pyridinilhydrazone.

Table 2 and FIG. 2 show the growth inhibition results for some specificcompounds representatives of the Formula Ia.

TABLE 2 Mycobacterium Smegmatis growth inhibition results Concen- % ofCelular Growth at 96 hours tration Compound Compound Compound (μM)Rifampicin 61 42 70 100.00 22.26% 24.45%  31.91% 38.9% 33.33 58.04%20.08%  41.52% 44.2% 11.11 90.62% 83.84%  98.17% 80.0% 3.70 81.40%69.76% 104.48% 96.6% 1.23 82.55% 75.35% 110.06% 95.9%

Example 4 Measurement of IC₅₀ Value

The inhibitory effect of a compound can be described by an IC₅₀ value,that is the concentration of inhibitor at which half (50%) inhibition ofthe maximal (100%) inhibition occurs. IC₅₀ values were determined bymeasuring the extent of inhibition over a range of concentrations of thecompounds of interest, preferably a range where the degree of inhibitionvaried from no inhibition (0%) to complete inhibition (100%). The IC₅₀value can be estimated from a plot of % inhibition against aconcentration of inhibitor, or can be calculated using data fittingprograms, such as IDBS XLfit™.

Based on the data obtained in the Mycobacterium smegmatis screeningassay, a curve fitting approach allowed for the determination ofinhibitory concentrations values such as the IC₅₀ that represents therequired concentration to inhibit 50% of the Mycobacterium smegmatisgrowth. The lower the IC₅₀, the higher the potency of the compounds toinhibit the Mycobacterium smegmatis. The IC₅₀ values obtained forspecific compounds under Formula Ia were depicted in Table 3:

TABLE 3 IC50 Values Compound IC₅₀ (μM) Rifampicin 24.82 Compound 6113.54 Compound 42 15.18 Compound 70 13.87

These results showed that the compounds representative of the presentinvention (compound n° 42 and its analogues, compounds n° 61 and 70)exhibit better IC₅₀ values than RMP which translate into more potencyfor Mycobacterium smegmatis inhibition.

As the results demonstrate surprisingly, the compounds representative ofthe present invention exhibit better IC₅₀ values and more potency thanthe active control, RMP, thereby confirming its superiority in terms ofinhibition of Mycobacterium smegmatis.

Lisbon, Nov. 19, 2013.

The invention claimed is:
 1. A compound represented by the followingFormula (Ib):

wherein R⁴ and R⁵ are each (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt or solvate, thereof.
 2. The compound according to claim1, wherein it is a pharmaceutically acceptable salt or solvate thereof.3. A pharmaceutical composition for use in the treatment and/orprevention of tuberculosis and related diseases, as an antimicrobialdrug, comprising: a compound according to claim 1; and apharmaceutically acceptable excipient, wherein the pharmaceuticallyacceptable excipient is at least one selected from the group consistingof binders, disintegrants, surfactants, glidants, lubricants,antioxidants, sequestrants, opacifiers or plasticizers.
 4. Thepharmaceutical composition according to claim 3, wherein tuberculosisand related diseases includes disease caused by Mycobacteriumtuberculosis Complex (MTbC), Mycobacterium leprae and nontuberculousmycobacteria (NTM).
 5. The pharmaceutical composition according to claim3, wherein the tuberculosis related disease is selected from the groupconsisting of primary tuberculosis disease, post-primary pleuropulmonarytuberculosis disease, post-primary extra-pulmonary tuberculosis diseaseinvolving at least one organ or system of a mammal.
 6. Thepharmaceutical composition according to claim 4, wherein diseases causedby Mycobacterium tuberculosis complex (MTbC) includes infection causedby organisms of the group selected from Mycobacterium tuberculosis(Mtb), Mycobacterium bovis, Mycobacterium africanum, Mycobacteriumcanetii and Mycobacterium microti.
 7. The pharmaceutical compositionaccording to claim 4, wherein diseases caused by non-tuberculousmycobacteria (NTM) includes infection caused by organisms of the groupselected from Mycobacterium avium complex (MAC), Mycobacteriumsmegmatis, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacteriumterrae, Mycobacterium scrofulaceum, Mycobacterium vaccae, Mycobacteriummarinum, Mycobacterium lentiflavum, Mycobacterium fortuitum,Mycobacterium chelonae, Mycobacterium abscessus, Mycobacteriumintracellulare and Mycobacterium avium.
 8. The pharmaceuticalcomposition according to claim 4, wherein the treatment groups arechildren or elderly patients.
 9. The pharmaceutical compositionaccording to claim 4, wherein the treatment group are patients with anyimmunosuppressive condition.
 10. The pharmaceutical compositionaccording to claim 4, wherein the composition is in the form of atablet, coated tablet, microcapsule, soft capsule, hard capsule, pellet,suppository, powder, solution, suspension, aerosol, syrup, drops, cream,paste, gel, ointment, tincture, lipstick or spray.
 11. Thepharmaceutical composition according to claim 4, wherein the treatmentcomprises administering the pharmaceutical composition to a mammal at adaily dosage of between 0.01 mg/kg body weight and 5000 mg/kg bodyweight.